AD represents a common progressive neurodegenerative disorder that is characterized neuropathologically by extracellular deposits composed of the amyloid-β (Aβ) protein and intra-cellular accumulation of phosphorylated tau protein. The story of successful discoveries in modern AD research using novel molecular biological tools started with the biochemical analysis of β-amyloid containing blood vessels (CAA, cerebral amyloid angiopathy) and amyloid plaques consisting of Aβ, which led to the isolation and sequencing of the gene encoding the larger amyloid precursor protein (APP) (Kang, et al. (1987) Nature 325, 733-736; Tanzi, et al. (1987) Science 235, 880-884).
For more than two decades, the amyloid hypothesis has been the central hypothesis in coining the molecular pathology of AD. According to this hypothesis, amyloid fibrils, which are large insoluble polymers of Aβ found in senile plaques, are the major trigger of neuron loss and dementia typical for AD. Albeit there are convincing genetic, biochemical and cell biological data for a major role of Aβ in AD, growing evidence points towards soluble Aβ oligomers rather than Aβ precipitated in plaques.
Haass and Selkoe have recently discussed the concept that soluble oligomers of Aβ as diffusible assemblies are capable of interfering with synaptic function and integrity providing an important opening for understanding the basis of memory loss in AD. They argued that small soluble oligomers affect synaptic structure and plasticity, whereas large, insoluble plaque deposits might function as reservoirs of the pathological oligomers (Haass and Selkoe (2007) Nat Rev Mol Cell Eliot 8, 101-112). Moreover, it has also been suggested that the extracellular formation of Aβ plaques and other AD pathological events are preceded by intraneuronal Aβ accumulation giving rise to a modified amyloid hypothesis.
Aβ, composed of mostly 1-40 or 1-42 amino acids, is a critical component in the etiology of the neurodegenerative AD. Aβ is prone to aggregate and forms amyloid fibrils progressively both in vitro and in vivo. To understand the process of amyloidogenesis, it is pivotal to examine the initial stages of the folding process. Recently, the equilibrium folding properties, assembly states, and stabilities of the early folding stages of Aβ1-40 and Aβ1-42 prior to fibril formation was examined. It was found that Aβ1-40 and Aβ1-42 have different conformations and assembly states upon refolding from their unfolded ensembles. Aβ1-40 is predominantly an unstable and collapsed monomeric species, whereas Aβ1-42 populates a stable structured trimeric or tetrameric species at concentrations above ˜12.5 μM. Thermodynamic analysis showed that the free energies of Aβ1-40 monomer and Aβ1-42 trimer/tetramer are ˜1.1 and ˜15/˜22 kcal/M, respectively. The early aggregation stages of Aβ1-40 and Aβ1-42 contain different solvent-exposed hydrophobic surfaces that are located at the sequences flanking its protease-resistant segment. It was concluded that the amyloidogenic folded structure of Aβ is important for the formation of spherical β oligomeric species.
Ono and colleagues (Ono, et al. (2009) Proc. Natl. Acad. Sci. 106, 14745-14750) clarified the ill-defined term “oligomere” because many different ones have been reported and often existing in rapid equilibrium with monomers and higher-order assemblies. They reported results of studies in which specific oligomers have been stabilized structurally, fractionated in pure form, and then studied using different biochemical, microscopic and neurotoxicity assays. Aβ1-42 monomers were largely unstructured, but oligomers exhibited order-dependent increases in β-sheet content. Dimerization and subsequent monomer addition are processes in which significant and asymmetric monomer conformational changes occur. Oligomer secondary structure and order correlated directly with fibril nucleation activity. Neurotoxic activity increased disproportionately (order dependence >1) with oligomer order. Their results provided significant insights into the biophysical and pathobiological behavior of Aβ, and importantly, into strategies for developing therapeutics for AD. The authors concluded that the “specific activity” of Ab1-42 assemblies depends nonlinearly on oligomer order. In fact, Ab1-42 dimers were reported to be 3-fold more toxic than monomers, and tetramers were 13-fold more toxic. The mechanism of toxicity is not well understood. Membrane permeabilization by amyloid oligomers may initiate a common group of downstream pathologic processes, including intracellular calcium dyshomeostasis, production of reactive oxygen species, altered signaling pathways, and mitochondrial dysfunction that represent key effectors of cellular dysfunction and cell death. Naturally secreted Aβ oligomers may directly impair synaptic function as has been shown to block hippocampal long-term potentiation (reviewed in Wirths et al. (2004) J Neurochem 91, 513-520).
Moreover, in vitro and in vivo analysis of amyloid deposits in AD revealed various N- and C-terminal variants. Increased C-terminal length of Aβ (from Abx-40 to Abx-42) in AD enhanced aggregation, early deposition and promoted the toxicity of Aβ. Beside Aβ peptides, starting with aspartate as the first amino acid (Aβ1), several N-truncated and modified Aβ species have been described. Among Aβ species present in AD plaques, it was reported that Aβ4-42 is a relatively abundant species in AD, aged controls and vascular dementia patients. Using immunoprecipitation in combination with mass spectrometry, Portelius and colleagues showed that Aβ1-40, Aβ1-42, pyroglutamate AβpE3-42 and Aβ4-42 are the dominant fractions in the hippocampus and cortex of AD patients. Interestingly, it has been demonstrated that N-terminal deletions enhance Aβ aggregation comparing Aβ1-42 with Aβ1-42.
Intra- or extracellular deposition of proteins is a feature of neurodegenerative diseases and serves as a molecular pathologic basis for classification as proteinopathies. While immunore-activity for Aβ or prion protein (PrP) is located predominantly extracellularly, proteins that deposit intracellularly include tau, α-synuclein, or TAR DNA Binding Protein 43 (TDP-43). Tauopathies include progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and argyrophilic grain disease (AGD), all predominated by the 4R isoform of the tau protein, and Pick's disease (PiD), a 3R tauopathy. Alpha-synucleinopathies comprise Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). TDP-43 proteinopathies include a group of frontotemporal lobar degenerations (FTLD-TDP), as well as sporadic amyotrophic lateral sclerosis (ALS) cases, while the most frequent form of prion disease is sporadic Creutzfeldt-Jakob disease (CJD). Kayed et al. have reported that different types of soluble amyloid oligomers display a common conformation-dependent structure that is unique to soluble oligomers regardless of sequence suggesting that they share a common mechanism of toxicity. These conformational antibodies against Aβ also recognized non-Aβ oligomers (Kayed et al. (2003) Science 300, 486-489).
One of the major problems in the amyloid hypothesis is the weak correlation between the severity of dementia and the density and localization of amyloid plaques in the brain of AD patients. Memory impairment and pathological changes in many AD mouse models occur well before the onset of plaque deposition. Soluble oligomers are low molecular weight non-fibrillar structures, which are stable in aqueous solution and remain soluble even after high speed centrifugation. Aβ oligomers develop preferentially intracellularly within neuronal processes and synapses rather than within the extracellular space. On the other side, Aβ can be taken up by certain cells, and then concentrated into endosomes/lysosomes. At high concentrations, vesicular Aβ aggregates form high molecular weight oligomers which are capable of seeding amyloid fibril growth. Results from several labs propose these oligomers to be the missing link in the amyloid hypothesis. While Aβ plaques are poor correlates for the clinical symptomatology in AD and Down syndrome patients, soluble oligomers are suggested to be good predictors for synaptic loss, neurofibrillary tangles and clinical phenotype. Just like in the human brain, studies using AD mouse models support the role of oligomers. In the Tg2576 mouse model, the appearance of Aβ dodecamers coincided with the onset of spatial memory impairment. Interestingly, injection of these purified oligomers into the ventricle of wildtype rats caused a dramatic drop in spatial memory performance. With regard to short-term effects, oligomers have been shown to impair synaptic plasticity by blocking LTP and reinforcing LTD. Others generated APP transgenic mice expressing the E693Δ mutation, which causes neuronal cell death and cognitive impairment by enhanced intracellular Aβ oligomerization without plaque formation.
Therefore, oligomers are thought to be a good target for therapeutic antibodies especially that this pool represents a minor subset (around 1.4% of total Aβ) if compared to the plaque pool which occupies the whole parenchyma. Reports have shown that monoclonal antibodies raised against oligomers prevent oligomer-induced toxicity, production of reactive oxygen species, and their attachment to synapses in primary hippocampal cells. In addition, Klyubin et al. have shown that immunization against Aβ oligomers neutralized LTP inhibition induced by Aβ oligomers injected in the hippocampus of rats (Klyubin et al. (2005) Nat Med 11, 556-561). Cell surface receptor clustering near or within synapses might be a mechanism for oligomer toxicity in AD. Aβ oligomers can exert their toxicity through binding at synapses. It has been demonstrated that oligomers undergo a progressive shift from an extrasynaptic, freely diffusive state toward the formation of static synaptic clusters acting as an extracellular scaffold for mGluR5.
Aβ dodecamers have been described using synthetic Aβ oligomers. Dimeric Aβ was purified from AD brains and also found to inhibit LTP in hippocampal slices and induce Tau hyperphosphorylation and other degenerative effects in cultured neurons. Selkoe and others reported that toxic Aβ oligomers to be primarily dimers and trimers of Aβ (Walshet al. (2000) Biochemistry 39, 10831-10839; Walsh et al. (2002) Nature 416, 535-539; Klyubin, et al. (2008) J. Neurosci. 28, 4231-4237).
Reviewed in detail by Hampel et al. (Hampel et al. (2010) Nat Rev Drug Discov 9, 560-574) discussed that AD biomarkers are needed to monitor drug safety, to identify individuals who are most likely to respond to specific treatments, to stratify presymptomatic patients and to quantify the benefits of treatments. Cerebrospinal fluid markers (for example, increased phosphophorylated tau and decreased Aβ1-42 levels) are helpful as trait markers of AD that have high sensitivity and specificity. However, they explained that these biomarkers have yet to show value as markers of disease state. Of interest, it has been reported that levels of Aβ1-42 are already fully decreased at least 5 to 10 years before conversion to AD dementia, whereas T-tau and P-tau seem to be later markers. Additionally, Aβ oligomers have also been studied as a potentially new biomarker in CSF. Analysis of HMW Aβ oligomers derived from synthetic Aβ1-42 by size-exclusion chromatography, revealed that using an ELISA specifically detected HMW Aβ oligomers of 40-200 kDa (Fukumoto et al. (2010) Faseb J 24, 2716-2726). The CSF levels of these HMW Aβ oligomers showed a negative correlation with Mini-Mental State Examination scores in AD and mild cognitive patients, which are presymptomatic for AD.
Analysis of amyloid deposits in AD brains revealed various N- and C-terminal variants. The increased C-terminal length of Aβ (from Aβx-40 to Aβx-42) enhances its aggregation properties. Faster aggregation leads to earlier Aβ deposition, which is believed to promote its toxicity. Recently, Aβ1-43 was discovered as a novel toxic peptide in AD. Besides Aβ peptides starting with aspartate as the first amino acid (Aβ1), several N-truncated and modified Aβ species have also been described. It was reported that Aβ4-42 is a relatively abundant species in AD, aged controls and vascular dementia patients. Using immunoprecipitation in combination with mass spectrometry, it was shown that Aβ1-40, Aβ1-42, AβpE3-42 and Aβ4-42 are the dominant fractions in the hippocampus and cortex of AD patients. Moreover, it has been demonstrated that N-terminal deletions enhance Aβ aggregation comparing Aβ4-42 with Aβ1-42. It was further demonstrated that Aβ1-42 and AβpE3-42 exhibited similar effects on neuronal cytotoxicity in primary cortical neurons and on memory impairment after intracerebroventricular injection in wildtype mice. AβpE3-42 is now an established factor contributing AD pathology and may even be aggravating the severity of the disease (Wittnam et al. (2012) Journal of Biological Chemistry 287, 8154-8162). While the possible role of Aβ4-42 although historically discovered first and found to be a major peptide in AD brain, its role as a biomarker or therapeutic target is not well explored.
The amyloid hypothesis in Alzheimer's disease (AD) considers amyloid β peptide (Aβ) deposition as the causative event triggering down-stream events like neurofibrillary tangles, cell loss, vascular damage and memory decline. A major complication of the amyloid hypothesis has been that so far none of the antibodies therapies directed against plaque-Aβ has revealed the expected outcome in clinical trials. Although plaque load has been successfully reduced, the cognitive decline could not be influenced. Moreover, in everyday clinical practice, Aβ1-42 ELISAs are used that do not discriminate between the different aggregation states (monomers up to high molecular weight oligomers (HMW)) and are lowered during the development of AD.
In view of the above, it is desirable to target all Aβx-42 low and high molecular weight oligomers specifically. The expected therapeutic benefit is to neutralize these oligomers by passive immunization as has been demonstrated recently for another oligomer-specific antibody 9D5 detecting only low molecular weight (LMW) AβpE3-42 (Wirths et al. (2010) J. Biol. Chem. 285, 41517-41524; and WO 2011/151076). Other antibodies specific for Aβ-peptides and certain oligomers are described in WO 2009/056490, U.S. Pat. No. 7,763,249, EP 2 210 901, and EP 2 246 427.
WO 2009/065054 discloses antibodies specific for the protofibril form of Aβ-peptides (˜670 kDA). However, these antibodies differ from the presently described ones, since the antibodies of WO 2009/065054 do not bind to trimeric or tetrameric human Aβ, i.e. low molecular weight forms of Aβ-oligomers (cf. FIGS. 1, 2A, and 3A of WO 2009/065054). Wang et al. (Wang et al. (2009) FEBS Letters 583, 579-584) disclose scFv's W8, W9, W20, and WC2, which bind to Aβ trimers, tetramers and little bigger oligomers. However, the exact epitope, and/or the sequence of the variable regions of these antibody molecules are not described.
Kim et al. (Kim et al. (2004) Neurobiology of Aging 25, S145) describe the synthesis of conformationally-defined Aβ haptens, which may be useful for a future selection of antibodies that can selectively sequester these folding intermediates.
Presently, it is an object of the invention to provide novel antibodies, which can be advantageously used in the treatment and diagnosis of AD.